Enzymes of Nitrogen Metabolism

Enzyme channeling is the directed transport of substrate intermediates between
active sites on an enzyme. Channeling increases the efficiency of catalysis tenfold
or more. Below we describe two enzymes from nitrogen metabolism that are well-characterized
examples of enzyme channeling.

TRYPTOPHAN SYNTHASE

Tryptophan synthase catalyzes the last two steps in the tryptophan synthesis
pathway. The first step generates indole (the double-ring portion of tryptophan's
side chain) from indole-3-glycerol phosphate (a molecule much smaller than its
name). In the second step, the substrate amino acid serine receives the indole,
which is added on to its side chain. This second step essentially transforms serine
into tryptophan.

The two active sites of tryptophan synthase

The two enzymatic activities of tryptophan synthase are found on two separate
subunits in bacteria, and on a homologous single polypeptide chain in yeast and
some other eukaryotes. The bacterial enzyme functions as a tetramer, (ab)2.
For simplicity, we'll use a single ab dimer from the
bacterium Salmonella as a model. Each subunit bears one active site (yellow);
they are about 25 Å apart.

Note that to see behind any surface rendering, you can use the Chime menu:
Select–Display List–Toggle Visibility (or Toggle Transparency).

Indole-3-glycerol phosphate (IGP), the substrate for the first reaction, binds
in the a subunit active site. Shown here is a structurally
similar inhibitor of the enzyme, indole propanol phosphate (IPP).

Indole propanol phosphate vs. indole-3-glycerol phosphate

Binding of a substrate to an enzyme usually triggers catalytic activity, making
it difficult to study enzyme structure with a bound substrate. One way to get
around this difficulty is to use an inhibitor of the enzyme. An inhibitor that
structurally resembles the substrate can bind correctly in the active site. The
small difference in structure, however, renders the enzyme unable to act—so
the enzyme can be studied with this bound "substrate analog." (It is then
important to perform additional studies to confirm that the structure of the enzyme
is not altered by the inhibitor, nor is it altered in the same way as when the
true substrate binds.)

The inhibitor indole propanol phosphate (IPP) is used to pinpoint and study
the active site of tryptophan synthase, which normally binds the substrate indole-3-glycerol
phosphate (IGP). Compare the two structures.

Review question 1

Production of indole, the rings of tryptophan

The first reaction, the conversion of indole-3-glycerol phosphate to indole,
is catalyzed by the a subunit of trytophan synthase.
Without indole-3-glycerol phosphate bound in the a
active site, a loop of the a subunit (Loop 6) flops
around, leaving the substrate binding site open. The structure of Loop 6 cannot
be determined while the loop is mobile. When indole-3-glycerol phosphate
binds in the a active site, a dramatic conformational
change occurs: Loop 6 (orange) is stabilized and covers the active site. This
traps the substrate and prevents it from diffusing away. When Loop 6 is unstable,
the enzyme is less active than when the loop is stabilized. Among other things,
this indicates that without this "lid," the substrate can be easily lost to the
surrounding solution.

Once indole-3-glycerol phosphate is bound in the a subunit, it is converted to indole by cleavage of the bond (flashing) between the glycerol phosphate moiety (white) and the indole moiety.

Serine plus indole makes tryptophan, with the help of a coenzyme

The second reaction takes place in the active site of the b
subunit. Here, a pyroxidal 5'-phosphate (PLP) prosthetic group temporarily forms
a covalent bond with the substrate serine. The pyroxidal 5'-phosphate holds the
serine in place for the addition of the indole rings that convert serine to tyrosine.
The rings are added in place of serine's side chain, which is an OH group (shown
as an oxygen atom since hydrogen atoms do not appear in X-ray crystallography).
Following the addition reaction, the bond linking tyrosine to the pyridoxal 5’-phosphate
group is cleaved, and tyrosine is released from the enzyme.

Have tunnel, will travel

It is 25 Å from the a active site to the b
active site. How does the indole intermediate make this long trip? Kinetic studies
show that the indole produced in the a subunit active
site reaches the b subunit active site in record time
(less than 1/1,000 of a second)! This and other evidence strongly suggests that
indole does not randomly diffuse to the b site, rather
it travels by an internal tunnel.

The protein has been slabbed approximately half-way through and some residues
have been removed in this view to make the tunnel visible. Experiment with
the slab to uncover and cover the inside of the protein (control–click–drag).

About tunnels in proteins

It is important to remember some of the fundamentals of molecular structure
when looking at a tunnel in a protein. Proteins are not static molecules, despite
their solid, motionless appearance. They vibrate, and flex, and some loops flop
about. As a result, any snapshot of a protein (such as an X-ray crystal structure)
only tells part of a story. Under some circumstances, tunnels are blocked. For
example, a few flexible side chains are known to move in and out of the tryptophan
synthase tunnel. So the structures of the tunnels shown in this tutorial may differ
slightly from their structures in the active enzymes.

The tunnel is conserved

The interiors of soluble proteins are generally packed solidly with nonpolar
residues. There are small cavities here and there, but the presence of a tunnel
of any appreciable length is highly unlikely to occur by chance. The fact that
the tunnel has survived natural selection (recall that tryptophan synthase is
conserved in bacteria and eukaryotes) strongly implies that the tunnel is necessary
for the function of the enzyme. In addition, the tunnel is predominantly lined
with hydrophobic groups (green), which are compatible with the aromatic indole
group that must pass through.

Tryptophan synthase is not the only enzyme that employs a channeling strategy. We now turn our attention to a particularly complex and exquisite enzyme, carbamoyl phosphate synthetase.

CARBAMOYL PHOSPHATE SYNTHETASE

Four reactions, three sites, two tunnels

Carbamoyl phosphate is a nitrogen-containing compound that is essential for
two critical metabolic pathways: the urea cycle and pyrimidine nucleotide synthesis.
Carbamoyl phosphate is built using the amino acid glutamine, bicarbonate (HCO3–),
and two molecules of ATP. The reactions also produce glutamate, ADP, and phosphate.

In E. coli, carbamoyl phosphate synthetase is a heterodimer of a large
and small subunit.

The enzyme has three active sites that are a considerable distance apart.

1)

The glutamine amidotransferase site removes ammonia (NH3)
from glutamine.This reaction requires the formation of a thioester bond between
the glutamine substrate and a cysteine residue in the active site. In this view,
the backbone of the glutamine substrate is at the opening of the site, and the
side chain extends deep into the binding pocket, where it has already formed a
thioester bond with cysteine. The glutamine NH3 group has already been
extracted.

Review question 2

Press "Display" to load the view for this question.

A thioester intermediate is formed by the covalent attachment of the glutamine
substrate to the catalytic cysteine. Can you identify the atom that is now in
the position previously occupied by the nitrogen of glutamine's NH3
group?

The carboxyphosphate site uses HCO3–
and ATP to make carboxyphosphate, which then reacts with the NH3 extracted
from glutamine to make a reactive compound called carbamate. ADP, which is a product
of this reaction, is shown in the active site.

3)

The carbamoyl phosphate site uses carbamate and a second
molecule of ATP to make carbamoyl phosphate.

Review question 3

Press "Display" to load the view for this question.

The third site also contains a product of the third reaction. Can you identify
the product shown in the active site. Enter the three letter abbreviation for
this compound and press "Submit:"

All three active sites of E. coli carbamoyl phosphate synthetase are
visible in the next display. Show the distance between any two sites by clicking
on an atom in or near a site, and then clicking on an atom in or near a second
site. You can read the distance in angstroms in the "picked atom" output
text in the title bar. You can also display the distance of these pairs of atom
clicks as a label directly on the molecule by toggling the distance monitor in
the Chime menu (Select–Mouse click Action–Toggle Distance Monitor).
Note that the substrates must travel from site 1 to site 2 and then on to site
3. Calculate the total distance the substrates must travel to produce carbamoyl
phosphate.

As you may have guessed by now, substrates traveling these distances by diffusing
from site to site could easily be lost, or, given the reactivity of the intermediates
in these reactions, they could react with other molecules to form compounds that
are useless for building carbamoyl phosphate. For example, NH3, the
product of the first reaction, readily extracts a proton from water to become
NH4+. Since the ammonium ion cannot react in site 2, the
substrate would be wasted. Similarly, the carbamate produced in reaction 2 is
a reactive molecule that could be lost to the surroundings. A channel, however,
could shelter these intermediates. Evidence strongly suggests that carbamoyl phosphate
synthetase, like tryptophan synthetase, channels substrates from site to site,
using two internal tunnels:

Tunnel 1:

Tunnel 2:

The residues that have been removed (faded out) to reveal the tunnels are those
that overlay them most directly. The yellow surface represents the far side of
the tunnel from the viewer, defining an approximate path between the sites.
Take the time to explore each tunnel by zooming (shift-drag), translating (Mac,
option-drag; PC, control–drag), and rotating the structure. Note that the
second tunnel is wider than the first. Why do you think this is so?

The presence of channels in multifunctional enzymes such as tryptophan synthase
and carbamoyl phosphate synthetase maximizes metabolic efficiency. As more enzyme
structures are solved, it seems likely that we will find more examples of tunnels
and other means of controlling the passage of intermediates.

To review the animation of intermediates moving through the tunnel of carbamoyl
phosphate synthetase, press the button below: